1. Field of the Invention
The present invention relates to a technology where stem cells from embryonic and adult brain are isolated, propagated, and differentiated efficiently in culture to generate large numbers of nerve cells. This technology, for the first time, enables one to generate large numbers of many different kinds of neurons found in a normal brain and provides a new foundation for gene therapy, cell therapy, novel growth factor screening, and drug screening for nervous system disorders.
2. Description of the Related Art
The brain is composed of highly diverse nerve cell types making specific interconnections and, once destroyed, the nerve cells (neurons) do not regenerate. In addition, the brain is protected by a blood-brain barrier that effectively blocks the flow of large molecules into the brain, rendering peripheral injection of potential growth factor drugs ineffective. Thus, a major challenge currently facing the biotechnology industry is to find an efficient mechanism for delivering potential gene therapy products directly into the brain in order to treat nervous system disorders.
Moreover, for a degenerative disease like Parkinson's, the most comprehensive approach to regain a lost neural function may be to replace the damaged cells with healthy cells, rather than just a single gene product. Thus, current and future success of gene therapy and cell therapy depends upon development of suitable cells that can (1) carry a healthy copy of a disease gene (i.e., a normal gene), (2) be transplanted into the brain, and (3) be integrated into the host's neural network. This development ideally requires cells of neuronal origin that (1) proliferate in culture to a large number, (2) are amenable to various methods of gene transfer, and (3) integrate and behave as the cells of a normal brain. However, there have been no-such cells for therapeutic purposes since neurons do not divide and therefore cannot be propagated in culture.
As alternatives, various transformed cells of neural and non-neural origins such as glias, fibroblasts, and even muscle cells, which can be proliferated in culture, have been used as possible vehicles for delivering a gene of interest into brain cells. However, such cells do not and cannot be expected to provide neuronal functions. Another alternative approach has been to force a neural cell of unknown origin to divide in culture by genetically modifying some of its properties, while still retaining some of its ability to become and function as a neuron. Although some "immortalized" cells can display certain features of a neuron, it is unclear whether these altered cells are truly a viable alternative for clinical purposes.
A developing fetal brain contains all of the cells germinal to the cells of an adult brain as well as all of the programs necessary to orchestrate them toward the final network of neurons. At early stages of development, the nervous system is populated by germinal cells from which all other cells, mainly neurons, astrocytes, and oligodendrocytes, derive during subsequent stages of development. Clearly, such germinal cells that are precursors of the normal brain development would be ideal for all gene-based and cell-based therapies if these germinal cells could be isolated, propagated, and differentiated into mature cell types.
The usefulness of the isolated primary cells for both basic research and for therapeutic application depends upon the extent to which the isolated cells resemble those in the brain. Just how many different kinds of precursor cells there are in the developing brain is unknown. However, several distinct cell types may exist:
a precursor to neuron only ("committed neuronal progenitor" or "neuroblast"), PA1 a precursor to oligodendrocyte only ("oligodendroblast"), PA1 a precursor to astrocyte only ("astroblast"), PA1 a bipotential precursor that can become either neuron or oligodendrocyte, neuron or astrocyte, and oligodendrocyte or astrocyte, and PA1 a multipotential precursor that maintains the capacity to differentiate into any one of the three cell types.
Fate mapping analysis and transplantation studies in vivo have shown that different neuronal types and non-neuronal cells can be derived from the same precursor cells.sup.1-5. In vitro analyses have also suggested that multipotential cells are present in the developing brain.sup.6, 7. Lineage analysis alone, however, does not directly identify the multipotential cells; nor does it define the mechanisms that drive them to different fates. Precursor cells from the central nervous system (CNS) have been expanded in vitro and differentiation into neurons and glia has been observed.sup.8-12 and, as detailed below, markedly different cell types have been obtained even when the culture conditions used were seemingly the same.
Because of the current lack of understanding of histogenesis during brain development, many investigators have used various terms loosely to describe the cells that they have studied, e.g., neuronal progenitor, neural precursor, neuroepithelial precursor, multipotential stem cell, etc. Thus, the nature of the cells so far described in the literature and culture conditions for obtaining them can only be compared to each other by their reported differentiation capacity. The entire subject of the isolation, characterization, and use of stem cells from the CNS has recently been reviewed.sup.33, 34, 38.
In summary, conditions have not been found to date, despite many reports, to successfully identify, propagate, and differentiate multipotential stem cells. A useful compilation of studies reporting culture of CNS precursor cells is found in Table 3, p. 172, of a recent review.sup.34 and further extended below.
Vicario-Abejon, C., Johe, K., Hazel, T., Collazo, D. & McKay, R., Functions of basic fibroblast growth factor and neurotrophins in the differentiation of hippocampal neurons, Neuron 15, 105-114 (1995).sup.12.
Cells expanded by Vicario-Abejon et al. are significantly different from those described in the present invention although the starting tissue (embryonic hippocampus), the mitogen (basic fibroblast growth factor, bFGF), and the basal medium (N2) are similar in both reports. Almost all of the cells expanded by Vicario-Abejon et al. failed to differentiate into any cell types but died in the absence of bFGF (as stated in the paper, pg. 106). This is also reflected in FIG. 3 of the paper where the number of MAP2 positive neurons is exceedingly low (50-100 cells out of an initial cell number of approximately 80,000 per well; i.e., far less than 1% in all reported conditions). Thus, differences in culture conditions, subtle as they may be, can yield cells with significantly different properties and this is, in fact, consistent with the main observation of the present invention that the extracellular environment can shift the developmental properties of the CNS stem cells.
Vicario-Abejon et al. used the following culture conditions which differ from the those described in the present invention:
1. Used enzymatic dissociation, 0.1-0.25% trypsin +0.4% DNAse I for the initial tissue dissociation as well as subsequent passaging. In the present invention, enzymatic dissociation effectively causes proteolyses of FGF receptors and causes cells to become unresponsive to bFGF and leads to differentiation.
2. Used 10% fetal bovine serum to stop the trypsin activity and to prime the cells from 4 hours to overnight before switching to serum free medium. In the present invention, serum even at less than 1% concentration shifts stem cells to astrocytic fate.
3. Cells were seeded at much higher density of 45,000 cells per cm.sup.2 and then grown to confluence before passaging by trypsin and serum. In the present invention, high cell density inhibits proliferation and causes spontaneous differentiation even in the presence of bFGF.
4. bFGF was given only intermittently every 2-3 days, and at 5 ng/ml, less than the optimal concentration disclosed in the present invention. This condition leads to partial differentiation of cells and subsequent heterogeneity of cell types in culture.
5. Basal medium consisting of "N2" components consisted of 5 ng/ml insulin, less than the optimal concentration disclosed in the present invention.
Ray, J.. Peterson, D., Schinstine, M. & Gage, F., Proliferation, differentiation, and long-term culture of primary hippocampal neurons, Proc. Natl. Acad. Sci. USA 90, 3602-3606 (1993).sup.10.
This study used culture conditions that are very similar to those described by Vicario-Abejon et al.--bFGF as the primary mitogen, serum-free medium, and E16 hippocampus. However, it reports isolation and expansion of a precursor population (neuroblasts) quite different from the cells of Vicario-Abejon et al. (undefined) as well as the multipotential stem cells described in the present invention. The reported cells had the following properties which markedly contrast from those of CNS stem cells:
1. The expanded cells under the reported condition are mitotic neurons with antigenic expressions of neurofilament, nestin, neuron-specific enolase, galactocerebroside, and MAP2 (Table I, p. 3604). The expanding CNS stem cells reported in the present invention express nestin, only, are negative for the above antigens, and are, therefore, a molecularly distinct population of cells from those described by Ray et al.
2. Ultrastructural analysis of the expanded cells in culture "demonstrated their histotypic neuronal morphology". The expanding CNS stem cells exhibit entirely different, non-neuronal morphology.
3. The mitotic "neurons" had a doubling time of 4 days and could be passaged and grown as continuous cell lines. The CNS stem cells double at every 20-24 hours and exhibit a characteristic regression of mitotic and differentiative capacity over time so that they cannot be maintained as stable cell lines indefinitely.
4. The culture system by Ray et al. generates "nearly pure neuronal cell cultures". The culture system in the present invention generates multipotential stem cells that can differentiate into all three major cell types of the brain, i.e., neurons, oligodendrocytes, and astrocytes.
Ray et al. used the following culture conditions which differ from those of the present invention.
1. Embryonic hippocampi were mechanically triturated without the use of an enzyme; however, cells were plated approximately 100,000 cells per cm.sup.2, optimal for neuronal survival, but almost 10 times higher cell density than optimal for expansion of CNS stem cells.
2. bFGF was given at 20 ng/ml, intermittently, at every 3-4 days.
3. Basal "N2" medium contained 5 .mu.g/ml insulin, less than optimal. Medium change was also prolonged at every 3-4 days.
4. Cells were passaged by using trypsin.
In conclusion, even seemingly small differences in culture conditions can result in isolation of vastly different cell types.
Ray. J. and Gage, F. H., Spinal cord neuroblasts proliferate in response to basic fibroblast growth factor, J. Neurosci. 14, 3548-3564 (1994).sup.39.
Ray and Gage report isolation and propagation of cells "that have already committed to a neuronal pathway are and expressing neuronal phenotypes (neuroblasts)" from spinal cord using bFGF. Again, although the primary mitogen is bFGF, their culture conditions are different and obtained cells markedly different from CNS stem cells.
1. E14-E16 spinal cord was used, a much later stage of development than optimal for stem cells.
2. The tissue was dissociated enzymatically by papain and DNase.
3. Initial plating was done in 10% fetal bovine serum.
4. There was a preliminary enrichment for a non-adherent cell population.
5. There was intermittent medium change and bFGF supplement, every 3-4 days.
Gage, F. H., Coates, P. W., Palmer, T. D., Kuhn, H. G., Fisher, L. J., Suhonen, J. O., Peterson, D. A., Suhr. S. T. & Ray, J., Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain, Proc. Natl. Acad. Sci. USA 92, 11879-11883 (1995).sup.35.
Gage et al. report isolation, propagation, and transplantation of cells from adult hippocampus. These mixtures of cells were maintained in culture for one year through multiple passages. 80% of them exhibit rather unusual properties such as co-expressing glial and neuronal antigens while remaining mitotic. These properties are not exhibited by stem cells isolated from the adult striatal subventricular zone.
Again, using bFGF as a primary mitogen, the authors derived markedly different cells than CNS stem cells reported in the present invention.
Gritti. A. et al., Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor, J. Neurosci. 16, 1091-1100 (1996).sup.40.
These authors report isolation and propagation of multipotential stem cells from the subventricular zone of adult brain by using bFGF. A significant difference in culture conditions used by Gritti et al. is that the cells are propagated as aggregated spheres without attachment to plate surface. Culture conditions by Gritti et al. require this aggregation of cells into spheres, using either bFGF or epidermal growth factor (EGF), as an essential step for propagating multipotential cells. This aggregation step alone essentially distinguishes the reported culture system from that of the present invention. The aggregation promotes undefined cell-cell interactions and results in uncontrollable differentiation/fate-shifts and overall in much less expansion and differentiation. Furthermore, this culture system and the result obtained by Gritti et al. are limited to adult brain where extremely small number of cells were obtained (10.sup.5 cells per brain) and have not been extended to various regions of embryonic brain.
The procedure in the present invention permits propagation of stem cells throughout the developing CNS as well as the striatum of the adult brain. It also uses adherent culture and actively avoids cell-cell contact and high cell density. As a result, it permits much more efficient expansion of the cells in an undifferentiated multipotential state and much more precise and efficient control over differentiation of the expanded cells.
Reynolds, B. & Weiss. S., Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system, Science 255, 1707-1710 (1992).sup.15.
Reynolds, B., Tetzlaff, W. & Weiss, S., A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J. Neurosci. 12, 4565-4574 (1992).sup.9.
Vescovi, A. L., Reynolds. B. A., Fraser. D. D., and Weiss, S., bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells, Neuron 11, 951-966 (1993).sup.41.
These three studies describe the original sphere cultures of neural precursor cells from adult and embryonic brain using EGF (epidermal growth factor). The expanded cells differentiate into neurons and astrocytes, but not into oligodendrocytes, and thus are thought to be a bipotential population, rather than multipotential. Another distinguishing property of the cells is that they respond only to EGF and not to bFGF in particular, whereas CNS stem cells respond similarly to both EGF and bFGF. Again, the sphere culture conditions are not comparable to those employed in the present invention because they require cell aggregation in which many additional undefined interactions are expected to occur.
Ahmed, S., Reynolds. B. A., and Weiss, S., BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors. J. Neurosci. 15, 5765-5778 (1995).sup.42.
This paper reports the effects of brain-derived growth factor (BDNF) on sphere cultures of embryonic neural precursor cells propagated with EGF. There is no further enhancement of the culture system per se.
Svendsen, C. N., Fawcett, J. W., Bentlage, C. & Dunnett, S. B., Increased survival of rat EGF-generated CNS Precursor cells using B27 supplemented medium. Exp. Brain Res. 102, 407-414 (1995).sup.36.
This study utilizes the sphere culture with EGF as described above to test a commercially available medium supplement called "B27". The study simply reports that use of B27 enhances cell survival (not neuronal survival) in a mixed culture containing neurons, astrocytes, and oligodendrocytes.
Kilpatrick, T. J. and Bartlett, P. F., Cloning and growth of multipotential neural precursors: requirements for proliferation and differentiation, Neuron 10, 255-265 (1993).sup.43.
The authors report existence of multipotential precursor cells in E10 mouse telencephalon by culturing single cells from the brain in bFGF plus serum. The results were based on 700 cells expanded clonally for 10 days, some of which, when differentiated in the presence of bFGF, serum, and astrocyte conditioned medium, could give rise to neurons. There was no mass expansion of the cells.
Kilpatrick, T. J. and Bartlett, P. F., Cloned multipotential precursors from the mouse cerebrum require FGF-2, whereas glial restricted precursors are stimulated with either FGF-2 or EGF, J. Neurosci. 15, 3653-3661 (1995).sup.44.
The authors utilize the clonal culture system reported in the above-described reference.sup.43 to test mitogenic efficacy of bFGF and EGF on cortical cells from E10 and E17 embryos. Again, the culture condition applies strictly to microculture in serum containing medium to demonstrate existence of different precursor cells in developing brain. There is no mass expansion, long-term culture, or systematic differentiation protocol.
Baetge, E. E., Neural stem cells for CNS transplantation, Ann. N.Y. Acad. Sci. 695, 285 (1993).sup.45.
This is a brief review paper summarizing various studies directed to isolating precursor cells and their derivatives in culture. It is somewhat outdated and most of the relevant original studies cited have been discussed above.
Bartlett. P. F. et al., Regulation of neural precursor differentiation in the embryonic and adult forebrain, Clin. Exp. Pharm. Physiol. 22, 559-562 (1995).sup.46.
This is also a brief review paper summarizing mostly previous works from the authors' laboratory in regard to their microculture studies where differentiation potentials of certain clones of precursors are tested in the presence of acidic FGF (aFGF), bFGF, serum, and/or astrocyte conditioned medium.
In addition, Sabate et al..sup.32 reported the culturing of a human neural progenitor with undefined differentiation capacity. Davis and Temple.sup.6 demonstrated the existence of multipotential stem cells in cortex by co-culturing with epithelial cells for short term (less than 100 cells altogether).
However, cell differentiation could not be controlled in any of the reported studies which precluded analysis of their lineage relations and the mechanisms regulating fate choice.
The present invention provides a method for efficiently propagating the undifferentiated germinal cells, i.e., stem cells of the central nervous system (CNS), in culture and defines conditions to effectively turn the undifferentiated cells into mature cell types. These undifferentiated cells or "CNS stem cells" display the multipotential capacity to differentiate into all three major cell types of a mature brain--neurons, astrocytes, and oligodendrocytes. Moreover, the same culture conditions enable isolation, expansion, and differentiation of equivalent multipotential cells from the adult brain.
Since the initial disclosure, additional reports have appeared. Most recent research on and use of CNS stem cells and neural progenitors have been further reviewed.sup.47-52. In addition, Reynolds and Weiss.sup.53 reported that embryonic striatal progenitors generated as spheres using EGF were able to differentiate into all three cell types including oligodendrocytes, astrocytes, and neurons. The frequency of EGF-responsive cells was limited to only 1% of the initial primary culture. Subcloning to establish self-renewal was questionable since up to 500 cells/well were used to generate the secondary "clones". Differentiation of the cells was induced by incorporating it serum in the medium. However, no data demonstrating all three cell types were presented from single-cell derived clones.
Weiss et al..sup.54 reported that multipotential CNS stem cells could be isolated from adult spinal cord and third and fourth ventricles by using a combination of EGF and bFGF but not with either alone.
Svendsen et al..sup.55 reported that neural precursor cells isolated from striatum and mesencephalon of 16 day old rat embryos (E16), when grafted into lesioned adult rat brains, failed to differentiate into neurons. They also reported that EGF-generated mesencephalon cells but not striatal cells differentiated into tyrosine hydroxylase (TH)-positive neurons, albeit in very low number (0.002%). There were no characterization of cells in vitro to ensure that the primary culture used contained no post-mitotic neurons carrying over from the tissue, especially given that the result could only be obtained with E16 tissue when most TH cells are already born.
Schinstine and Iacovitti.sup.56 reported that some of the astrocytes derived from EGF-generated neural precursor cells expressed neuronal antigens such as tau and MAP2. Qian et al..sup.57 reported that different concentrations of bFGF proliferate stem-like cells of E10 mouse cortex with varying differentiation potentials ranging from only neuronal to multipotential.
Palmer et al..sup.65 reported that multipotential CNS stem cells could be isolated from adult rat hippocampus. 84% of the cells they expanded, however, co-expressed MAP2c and O4, immature neuronal and oligodendroglial markers. Only 0.2% were MAP2ab positive and less than 0.01% were positive for other neuronal markers such as tau and neurofilament 200. Such properties are quite different from the properties described in the Examples in the present application.
Finley et al..sup.66 reported that the mouse embryonic carcinoma cells line, P19, can form neuronal polarity and be eletrophysiologically active when induced by retinoic acid and serum. Strubing et al..sup.67 reported that embryonic stem cells grown in serum-containing medium could differentiate into electrophysiologically active neurons in vitro. Okabe et al..sup.68 also reported differentiation of some of embryonic stem cells into neurons in vitro.
Gritti et al..sup.40 reported that multipotential stem cells could be isolated from adult mouse subependyme by EGF and bFGF, which when differentiated, could be eletrophysiologically active and express GABA-, gluatamate-, and ChAT-immunoreactivities, but not others. The frequency of such neurons, however, was not documented and thus it is difficult to ascertain how efficient neuronal maturation was. Moreover, these neuronal phenotypes derived from dividing stem cells were not directly demonstrated by BrdU labeling. This is particularly relevant since aggregate cultures are extremely prone to be contaminated by primary neurons from the tissue, which carry over for several passages. Weiss et al..sup.49, in fact, stated that only GABA-positive cells could be obtained from their cultures. Most of the GABA-positive cells may be oligodendrocytes.
Feldman et al..sup.69 reported electrophysiological studies of EGF-generated rat neural precursors. They found that most, if not all, electrophysiologically active cells are in fact non-neuronal, and that glial cells do contain voltage-sensitive Na channels that evoke action potential-like conductances.
Results such as these illustrate that identifying CNS stem cells, defining conditions that stably maintain CNS stem cell properties for long-term, and controlling their differentiation into mature cell types are neither obvious nor predictable to those skilled in this art.